Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
  • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
  • C12P7/625—Polyesters of hydroxy carboxylic acids
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/829—Alcaligenes

Definitions

• This invention relates to a process for the production of HV/HB copolymers and for microorganisms for use in such a process.
• strains of the species Rhodococcus capable of producing, and accumulating, HV/HB copolymers have the characteristic that they overproduce at least one amino acid, specifically at least one of methionine and preferably isoieucine or valine, and that these overproduced amino acids are metabolised to propionyl CoA, and thence to the required HV monomer units.
• microorganisms capable of producing, and accumulating, HV/HB copolymers which comprises providing within a suitable microorganism the ability to overproduce at least one of isoleucine, methionine and valine. Such microorganisms may then be used in a general microbiclogical process for the production of HV/HB copolymers, when provided with suitable substrate for example comprising glucose, or a compound metabolisable to glucose.
• the invention comprises a method of producing a microorganism capable of sythesizing HV/HB copolymer when cultivated on a substrate containing a component from which the microorganism can synthesise both HV and HB monomer units from a first microorganism which is capable of metabolising the substrate to acetyl CoA and is also capable of degrading an amino acid selected from methionine, valine and isoleucine to propionyl CoA which method comprises when said first microorganism is capable of synthesising and polymerising HB monomer units when cultivated on said substrate but is not capable of amino acid overproduction, inducing overproduction of said amino acid in said first microorganism, and when said first microorganism is not capable of synthesising and polymerising HB monomer units when cultivated on said substrate but is capable of overproducing said amino acid, transferring to said first microorganism the genetic information necessary for it to synthesise and polymerise HB monomer units when cultivated on said substrate from
• the microorganism is preferably a strain of Alcaligenes.
• Alcaligenes eutrophus NCIMB 11599, and NICMB 40124 are examples of such strains.
• NCIMB 40124 is preferred in that in this strain the use of propionyl CoA for growth is blocked, thereby giving rise to improved efficiency of HV/HB copolymer production, and also higher percentages of HV monomer in the copolymer.
• the method for preparing microorganisms in which amino acid overproduction is apparent may include exposure of the microorganism to amino acid analogues such as amino butyric acid, and norvaline.
• amino acid analogues such as amino butyric acid, and norvaline.
• the required mutations may be spontaneous, or may be induced by the use of suitable mutagenic agents such as UV light, ethane methane sulphonate and N-nitroso-guanidine.
• the microorganism produced according to the method of the present invention may be cultivated, in the general microbiological process, on a substrate in which the component from which the monomer units are synthesised is glucose, or a compound metabolisable to glucose.
• the component is one or more of glucose, fructose, acetate, lactate, ethanol or derivatives thereof.
• Alcaligenes eutrophus strain NCIMB 40124 was used as the initial strain which, by a mutation process, gave rise to an amino acid overproducing mutant.
• Alcaligenes eutrophus NCIMB 40124 was grown on glucose mineral salts medium for approximately 16 hours at 30° C., until an optical density of greater than 1 at 640 ⁇ m was reached.
• the subcultures of the colonies for Example 1 were cultivated on typical media which contained sources of nitrogen (ammonium salts), carbon (glucose), and trace elements such as phosphate, sulphate and magnesium.
• the cultivation was allowed to proceed to nutrient limitation, specifically nitrogen limitation as used in established HB copolymer production. Those cultures which accumulated HV/HB copolymer were regarded as positive. In particular Alcaligenes eutrophus strain PS-101 was isolated.
• Alcaligenes eutrophus strain PS-101 was further cultivated under copolymer accumulating conditions on a medium in which glucose was the sole carbon source.
• Example 3 was repeated except that Alcaligenes eutrophus strain NCIMB 40124 was used instead of mutant strain PS-101.
• Strain NCIMB 40386 was produced from Alcaligenes eutrophus strain NCIMB 40124 by the following means:
• a single colony of NCIMB 40124 was inoculated into the medium listed in Table 1 containing 1% glucose.
• the inoculated medium was incubated by shaking at 180 rpm and at a temperature of 28° C. until the mid logarithmic phase.
• the cells were harvested by centrifugation, washed with 0.85% sodium chloride solution and re-suspended in 0.85% sodium chloride solution and transferred to a glass petri dish.
• the cells were exposed to UV light to achieve a 99% kill and then inoculated into the medium listed in Table 1 containing 1% glucose.
• the inoculated medium was incubated by shaking at 180 rpm and at 28° C. for 16 hours in the dark to allow expression of the mutations.
• the resulting population was spread onto agar plates containing the medium listed in Table 1 and 500 ⁇ g/ml ⁇ - amino-butyric acid. The plates were incubated for seven days at 28° C. Resistant colonies were selected which showed halos of satellite growth: the halo indicated that the resistant colonies were excreting a substance which relieved the inhibition for the surrounding sensitive cells. This selection produced strain NCIMB 40386 with the ability to produce and accumulate HB/HV copolymer when provided with glucose as shown by Example 6.
• the growth medium listed in Table 1 containing 1% glucose and 500 ⁇ g/ml ⁇ - aminobutyric acid was inoculated with a culture of Alcaligenes eutrophus NCIMB 40386 produced as described in Example 5.
• the inoculated medium was shaken using a stirrer speed of 180 rpm and (2") approximately 50 mm (2 inches) throw at a temperature of 28° C. for 24 hours.
• the cells were harvested by centrifugation, washed with 0.85% sodium chloride solution and then resuspended in the growth medium listed in Table 1 containing 1% glucose and replacing the phosphate buffer with 0.5 m Hepes buffer.
• the medium was incubated shaking at 180 rpm, (2") approximately 50 mm (2 inches) throw, at a temperature of 28° C. for 48 hours.
• the cells were harvested by centrifugation, washed with 0.85% sodium chloride solution and freeze dried.
• the freeze dried cells were ground to a fine powder with a pestle and mortar. 10 g of cells were refluxed with 500 mls chloroform for one hour.
• the resulting solution was filtered under vacuum and the polymer was recovered by precipitation with methanol and water.
• the polymer solution was slowly added, with mixing to a solution+consisting of 2 litres water and 500 mls distilled water.
• the polymer precipitate was then filtered and dried for 24 hours at 80° C.
• Freeze dried cells were prepared for analysis using High Performance Liquid Chromatography (HPLC) by extraction in 55% perchloric acid and neutralised with 20% potassium hydroxide.
• HPLC High Performance Liquid Chromatography
• Alcaligenes eutrophus strain H16 a widely available micro-organism available for example from NCIMB as NCIMB 10442 was used as the initial strain in a mutation process which gave rise to amino acid overproducing mutants.
• Algaligenes eutrophus strain H16 was grown on mineral salts medium containing 1% fructose and the resultant cells subjected to UV mutagenesis by exposure to sufficient UV dose to give between 90 to 99% kill.
• the survivors were inoculated into nutrient broth (0.8% Oxoid) and were shaken whilst being incubated overnight in the dark at a temperature of 28° C. to allow expression of the mutations.
• the colistine technique was used to enrich the population of mutants for auxotrophs.
• Cells were incubated in the presence of 50 ⁇ g.ml -1 colistine for 10 hours. Dilutions of the survivors were plated onto complete medium agar (0.8% nutrient broth and 01.5% NaCl).
• Replica plating to fructose mineral salts agar was used to identify auxotrophic colonies. By this means the mutant Alcaligenes eutrophus H16-G131 was isolated. The mutant was found to require isoleucine (Reh M and Schlegel H G. 1969. Arch. Mikrobiol. 67. 99-109).
• Colonies of Alcaligenes eutrophus H16-R3 were inocculated into 10 mls of nutrient broth (oxoid) and a shake flask preculture was incubated overnight at 30° C. This preculture was then used to inoculate 300 mls of 1% fructose-mineral salts medium containing only 0.05% ammonium chloride in a 1 liter flask. The flask was incubated with shaking at 30° C.
• PHA polyhydroxyalkanoate
• NCIMB means NCIMB Limited, 23 Machar Drive, Aberdeen, Scotland, UK AB2 1RY. NCIMB 40386 was deposited on Mar. 20, 1991 with NCIMB.
• Alcaligenes eutrophus H16-R3 is deposited as DSM 6525 with Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSM), Mascheroder Weg 1b, D-3300 Braunschweig, Germany on May 15, 1991.
• DSM Deutsche Sammlung von Mikroorganismen and Zellkulturen

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• 1990
  • 1990-05-25 GB GB909011777A patent/GB9011777D0/en active Pending
• 1991
  • 1991-05-24 WO PCT/EP1991/000978 patent/WO1991018995A1/en not_active Ceased
  • 1991-05-24 CA CA002083695A patent/CA2083695C/en not_active Expired - Lifetime
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  • 1991-05-24 EP EP91910223A patent/EP0535012B1/de not_active Expired - Lifetime
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